Studies On Orthogonal Separation And Purification Of Anti-cancer Components From Toad Skin

As a main resource of bufadienolides, Bufo bufo gargarizans Cantor (toad skin) is a well-known traditional medicine in Asian and its preparations are now widely used in clinical therapy for heart diseases and various cancers in China. However, as a biological sample, the components of toad skin extract are complex and the separation and purification of active compounds is also a tough work. In this paper, based on the novel homedmade chromatographic materials, three two-dimension high performance liquid chromatography (2D-HPLC) methods were developed for the separation and purification of actived components (bufadienolides and nitrogen polar compounds) according to their different structures. In addition, a new method was developed for the synthesis and separation of the total bufogenins-3-O-sulfonate.A unique method based on a homemade polar-modified C18stationary phase was developed and successfully applied to separate polar compounds from the toad skin. Compared with previous purification systems based on the C18stationary phase, the poor retention problem of polar compounds was well resolved by using of the polar-modified C18column and good resolutions were achieved as well. Seven compounds were purified and five of them (uracil, hypoxanthine,3-Hydroxy-4H-Pyrazolo [4,5-d] Pyridazin-7(1H)-One, thymine, bufothionine) were identified by MS,’H-NMR and13C-NMR. Additionally,3-Hydroxy-4H-Pyrazolo [4,5-d] Pyridazin-7(1H)-One was identified as a new compound and hypoxanthine was found from the skin of Bufo bufo gargarizans Cantor for the first time.We reported a new HPLC method based on positively charged C18material (XCharge C18) at low pH, which utilized the distinct ionic feature of the amino acid conjugated bufadienolides (AACBs) and the free form bufadienolides (AAUBs). Compared with the conventional C18, the peak tailing problem of AACBs was successfully solved and better resolutions were achieved on the XCharge C18. In addition, the application of this method in other fractions was also validated. Taking one fraction as an example, the method was validated by liquid chromatography-mass spectrometry (LC-MS), and then4AACBs as well as4AAUBs were simultaneously purified by a preparative XCharge C18column.A class separation approach based on the hydrophilic Click TE-Cys cartridge was developed to help solve the co-elution problem of AACBs and AAUBs on the conventional C18column according to their distinct hydrophilic properties. Using the excellent orthogonal hydrophilic solid-phase extraction method, AACBs and AAUBs were selectively divided into two groups, and the co-elution problem was successfully resolved. Twelve high-pure compounds were obtained from one active fraction from toad skin via prep-HPLC. By this novel class separation method, the poor orthogonality in the2D-RPLC×RPLC system was well resolved, and this will accelerate the process of active compound discovery.In this paper, we firstly took the arenobufagin as an example to develop an effective method for the synthesis and separation of arenobufagin-3-O-sulfonate. In addition, the total bufogenins were sulfonated and separated by this method. A large number of potential anticancer new compounds were obtained, which provided lots of agents for further screening to achieve high effective and low toxic compounds.